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ISSN 1672-9145                                                 Acta Biochim Biophys Sin 2004, 36(9): 609–617                                                     CN 31-1940/Q

Cleavage of Supercoiled Circular Double-stranded DNA Induced by a Eukaryotic Cambialistic Superoxide Dismutase from Cinnamomum camphora

 

 

Bao-Zhong WANG, Xu-Bin WEI&, and Wang-Yi LIU*

 

State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

 

Abstract        A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity requires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random.

 

Key words        cambialistic SOD; Fenton-type reaction; hydroxyl radical; relaxed circular duplex DNA; supercoiled DNA cleavage

 

 

 

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Received: Mar 17, 2004         Accepted: July 9, 2004

This work was supported by grants from Academia Sinica (39570173) and the National Natural Science Foundation of China (39870186)

&A research student from the Institute of life science, Lanzhou University

*Corresponding author: Tel, 86-21-54921238; Fax, 86-21-54921011; E-mail, [email protected]